Degradation and endoplasmic reticulum retention of unassembled alpha- and beta-subunits of Na,K-ATPase correlate with interaction of BiP.

Assembly of alpha- and beta-subunits in the endoplasmic reticulum is a prerequisite for the structural and functional maturation of oligomeric P-type ATPases. In Xenopus oocytes, overexpressed, unassembled alpha- and beta-subunits of Xenopus Na,K-ATPase are retained in the endoplasmic reticulum (ER) and are degraded with different kinetics, while unassembled beta-subunits of gastric H, K-ATPase leave the ER. In this study, we have investigated the role of the immunoglobulin-binding protein, BiP, in the folding, assembly, and ER retention of ATPase subunits. We determined the primary sequence of Xenopus BiP and used polyclonal antibodies to examine the interaction with BiP of various wild type and mutant alpha- and beta-subunits overexpressed in Xenopus oocytes. Our results show that ER-retained, unassembled Na,K-ATPase beta-subunits, but not transport-competent H,K-ATPase beta-subunits, efficiently associate with BiP until assembly with alpha-subunits occurs. Furthermore, the kinetics of BiP interaction with unassembled wild type and with mutant Na,K-ATPase beta-subunits parallels their respective stability against cellular degradation. Finally, alpha-subunits that are overexpressed in oocytes and are rapidly degraded and endogenous oocyte alpha-subunits that are stably expressed as individual assembly-competent proteins also interact with oocyte or exogenous BiP, and the interaction time correlates with the protein's stability. These data demonstrate for the first time that BiP might be involved in a long term maturation arrest and/or in the ER quality control of a multimembrane-spanning protein and lend support for a universal chaperone function of BiP.